Targeted Protein Degradation via Lysosomes.

In the scope of targeted protein degradation (TPD), proteolysis-targeting chimeras (PROTACs), leveraging the ubiquitin-proteasome system, have been extensively studied. However, they are limited to the degradation of soluble and membrane proteins, excluding the aggregated and extracellular proteins and dysfunctional organelles. As an alternative protein degradation pathway, lysosomes serve as a feasible tool for accessing these untouched proteins and/or organelles by proteosomes. Here, we focus on reviewing the emerging lysosome-mediated TPD, such as AUTAC, ATTEC, AUTOTAC, LYTAC, and MoDE-A. Intracellular targets, such as soluble and aggregated proteins and organelles, can be degraded via the autophagy-lysosome pathway. Extracellular targets, such as membrane proteins, and secreted extracellular proteins can be degraded via the endosome-lysosome pathway. In addition, we summarize the mechanism and regulation of autophagy, available methods and assays for monitoring the autophagy process, and the recently developed chemical probes for perturbing the autophagy pathways.

Applications of covalent chemistry in targeted protein degradation.

Proteolysis-targeting chimeras (PROTACs) and targeted covalent inhibitors (TCIs) are currently two exciting strategies in the fields of chemical biology and drug discovery. Extensive research in these two fields has been conducted, and significant progress in these fields has resulted in many clinical candidates, some of which have been approved by FDA. Recently, a novel concept termed covalent PROTACs that combine these two strategies has emerged and gained an increasing interest in the past several years. Herein, we briefly review and highlight the mechanism and advantages of TCIs and PROTACs, respectively, and the recent development of covalent PROTACs using irreversible and reversible covalent chemistry.

Potential use of synthetic α-galactosyl-containing glycotopes of the parasite Trypanosoma cruzi as diagnostic antigens for Chagas disease.

A synthetic glycoarray containing non-reducing α-galactopyranosyl moieties related to mucin O-glycans of the parasite Trypanosoma cruzi was evaluated by a chemiluminescent enzyme-linked immunosorbent assay with sera from patients with chronic Chagas disease. Our data revealed the disaccharide Galα(1,3)Galß as the immunodominant glycotope, which may eventually be employed as a diagnostic antigen for Chagas disease.

Quantification of glutathione with high throughput live-cell imaging.

Reduction oxidation (redox) reactions are central in life and altered redox state is associated with a spectrum of human diseases. Glutathione (GSH) is the most abundant antioxidant in eukaryotic cells and plays critical roles in maintaining redox homeostasis. Thus, measuring intracellular GSH level is an important method to assess the redox state of organism. The currently available GSH probes are based on irreversible chemical reactions with glutathione and can't monitor the real-time glutathione dynamics. Our group developed the first reversible reaction based fluorescent probe for glutathione, which can measure glutathione levels at high resolution using a confocal microscope and in the bulk scale with a flow cytometry. Most importantly it can quantitatively monitor the real-time GSH dynamics in living cells. Using the 2 nd generation of GSH probe, RealThiol (RT), this study measured the GSH level in living Hela cells after treatment with varying concentrations of DL-Buthionine sulfoximine (BSO) which inhibits GSH synthesis, using a high throughput imaging system, Cytation™ 5 cell imaging reader. The results revealed that GSH probe RT at the concentration of 2.0 µM accurately monitored the BSO treatment effect on GSH level in the Hela cells. The present results demonstrated that the GSH probe RT is sensitive and precise in GSH measurement in living cells at a high throughput imaging platform and has the potential to be applied to any cell lines.

Discovery of a potent BTK and IKZF1/3 triple degrader through reversible covalent BTK PROTAC development.

Building on our previous work on ibrutinib-based reversible covalent Bruton's tyrosine kinase (BTK) PROTACs, we explored a different irreversible BTK inhibitor poseltinib as the BTK binder for PROTAC development. Different from ibrutinib, converting the irreversible cysteine reacting acrylamide group of poseltinib to a reversible covalent cyano-acrylamide group dramatically decreases the binding affinity to BTK by over 700 folds. Interestingly, one of the reversible covalent BTK PROTACs based on poseltinib with a rigid linker, dubbed as PS-RC-1, is highly potent (IC50 = ~10 nM) in Mino cells but not in other mantle cell lymphoma (MCL) cell lines, such as Jeko-1 and Rec-R cells. We showed that PS-RC-1 potently induces degradation of IKZF1 and IKZF3 but not BTK or GSPT1, accounting for its toxicity in Mino cells. We further decreased the molecular size of PS-RC-1 by shrinking the BTK binding moiety and developed PS-2 as a potent BTK and IKZF1/3 triple degrader with high specificity.

The odorant receptor co-receptor from the bed bug, Cimex lectularius L.

Recently, the bed bug, Cimex lectularius L. has re-emerged as a serious and growing problem in many parts of the world. Presence of resistant bed bugs and the difficulty to eliminate them has renewed interest in alternative control tactics. Similar to other haematophagous arthropods, bed bugs rely on their olfactory system to detect semiochemicals in the environment. Previous studies have morphologically characterized olfactory organs of bed bugs' antenna and have physiologically evaluated the responses of olfactory receptor neurons (ORNs) to host-derived chemicals. To date, odorant binding proteins (OBPs) and odorant receptors (ORs) associated with these olfaction processes have not been studied in bed bugs. Chemoreception in insects requires formation of heteromeric complexes of ORs and a universal OR coreceptor (Orco). Orco is the constant chain of every odorant receptor in insects and is critical for insect olfaction but does not directly bind to odorants. Orco agonists and antagonists have been suggested as high-value targets for the development of novel insect repellents. In this study, we have performed RNAseq of bed bug sensory organs and identified several odorant receptors as well as Orco. We characterized Orco expression and investigated the effect of chemicals targeting Orco on bed bug behavior and reproduction. We have identified partial cDNAs of six C. lectularius OBPs and 16 ORs. Full length bed bug Orco was cloned and sequenced. Orco is widely expressed in different parts of the bed bug including OR neurons and spermatozoa. Treatment of bed bugs with the agonist VUAA1 changed bed bug pheromone-induced aggregation behavior and inactivated spermatozoa. We have described and characterized for the first time OBPs, ORs and Orco in bed bugs. Given the importance of these molecules in chemoreception of this insect they are interesting targets for the development of novel insect behavior modifiers.